The invention relates to isolation and culture of porcine hepatocytes, useful, e.g., in liver assist devices.
The primary requirement of cells in a liver support system, e.g., a liver assist device (LAD), is the preservation of the in vivo metabolic functions that prevent and/or decrease the progress of hepatic encephalopathy (HE) in patients with acute liver failure. While the precise pathogenesis of this syndrome remains unknown, endogenous benzodiazepine-like substances (Basile et al., 1991, Pharmacol. Rev. 43:27-71) and ammonia (Butterworth et al., 1987, Neurochemical Pathology 6:1-12) have both been identified as important factors in its development. Benzodiazepines, as well as other drugs or toxins, are metabolized by the liver by a family of enzymes that catalyze their oxidative degradation (phase I metabolism), which, in turn, facilitates further detoxification steps (phase II metabolism). The oxidative enzymes, known collectively as the P450 system, can decay very rapidly in cultured hepatocytes (Reid et al., 1984, Hepatology 4:548-559) and are partially or poorly expressed in liver cell lines (Clayton et al., 1985, Mol. Cell. Biol. 5:2633-2641). Cultured hepatocytes have also been reported to lose function and ultimately die over the first few days in culture (Reid et al., supra).
Since healthy human liver cells are seldom available in adequate numbers to support clinical LAD use, hepatoma cells (malignant and transformed hepatocytes) (Wolf et al., 1975, Trans. Amer. Soc. Artif. Int. Organs 21:16-26), have been used to seed the intercapillary space of hollow fiber bioreactors. Mouse liver cells (Hager et al., 1983, ASAIO 6:26-35) and rat hepatocytes (Jauregui et al., 1989, In Butterworth et al., eds., Hepatic Encephalopthy, New Jersey: The Humana Press, pp. 339-351) have also been used to seed LAD devices. While rodent hepatocytes offer a model for preliminary LAD research, the limited number of liver cells available per animal prohibits a direct scale up to human devices. Primary porcine hepatocytes (Rozga et al., 1993, Ann. Surg. 217:502-511; Rozga et al., 1994, Ann. Surg. 219:538-546) have recently been used in LADs; however primary hepatocytes are difficult to proliferate in vitro (Enat et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:1411-1415) and lose phenotypic expression in culture (Reid et al., supra).